The wide functional diversity of proteins catalysis, regulation, transport, and structure has made them desirable bioresponsive molecules for integration into materials and medicine. Protein-polymer bioconjugates have already shown an impressive range of altered or improved properties (see Borner et al., J. Polym. Sci. Part a-Polym. Chem. 2010, 48, 1; Depp et al., Acta Biomat. 2009, 5, 560; Gao et al., Proc. Nat. Acad. Sci. USA 2009, 106, 15231; Krishna et al., Biopolymers 2010, 94, 32; Lutz et al., Prog. Polym. Sci. 2008, 33, 1; Nicolas et al., Macromol. Rapid Comm. 2007, 28, 1083; Connor et al., Polym. Rev. 2007, 47, 9). Protein-polymer bioconjugates also have shown efficient pharmacokinetics and therapeutic potency (see Gao et al.; Krishna et al.; Lutz et al.; Nicolas et al.; Lele et al., Biomacromolecules 2005, 6, 3380).
Protein polymers bioconjugates have been prepared in two general ways: either by graft-to methods where a preformed functionalized polymer is attached to an amino acid, cofactor or end group, or by the graft-from method where a location on the purified protein is functionalized with an initiator and then the polymer is grown from that site (see Krishna et al.; Liu et al., Ang. Chem., Int. Ed. 2007, 46, 3099; Zeng et al., Chem. Comm. 2007, 1453; Heredia et al., J. Am. Chem. Soc. 2005, 127, 16955. Thus far, the graft-from methods employed for residue-specific incorporation of polymerization initiators into proteins are limited to the N-terminal position or specific natural amino-acid directed linkages (see Depp et al.; Gao et al.; Le Droumaguet et al., Ang. Chem., Int. Ed. 2008, 47, 6263; Lele et al.; Canalle et al., Chem. Soc. Rev. 2010, 39, 329. Both methods suffer from challenging purification of intermediates and/or the inability to efficiently control the number or location of polymer connections to the protein, which compromises protein structural integrity. While the many graft-to and graft-from experiments using natural amino acids on proteins have illustrated the immense potential impact of well-defined protein-polymer conjugates, their application is limited by technical shortcomings associated with their synthesis and purification.
Previous protein-polymers have been prepared using an atom transfer radical polymerization (ATRP) technique (see Wang et al., Am. Chem. Soc. 1995, 117, 5614; Matyjaszewski & Xia, Chem. Rev. 2001, 101, 2921; Matyjaszewski & Tsarevsky, Nature Chem. 2009, 1, 276) wherein an ATRP initiator attached to the protein provides a linkage between the protein and growing polymer chain.
Atom-transfer radical polymerization (ATRP) and other controlled/living radical polymerization (CRP) methodologies including nitroxide mediated polymerization (NMP) and reversible addition fragmentation transfer (RAFT) systems allow unprecedented control over polymer dimensions (molecular weight), uniformity (polydispersity), topology (geometry), composition and functionality. [Matyjaszewski, K., Ed. Controlled Radical Polymerization; ACS: Washington, D.C., 1998; ACS Symposium Series 685. Matyjaszewski, K., Ed.; Controlled/Living Radical Polymerization. Progress in ATRP, NMP, and RAFT; ACS: Washington, D.C., 2000; ACS Symposium Series 768; Matyjaszewski, K., Davis, T. P., Eds. Handbook of Radical Polymerization; Wiley: Hoboken, 2002; Qiu, J.; Charleux, B.; Matyjaszewski, K. Prog. Polym. Sci. 2001, 26, 2083; Davis, K. A.; Matyjaszewski, K. Adv. Polym. Sci. 2002, 159, 1.] ATRP is a controlled radical polymerization (CRP) technique; therefore, monomers and cross-linkers may be incorporated in a predictable, controlled, and programmed manner to yield polymer chains of essentially equal length, as defined by the ratio of consumed monomer to the added initiator. Moreover, the functionality present on the introduced initiator is preserved and forms both the α- and ω-chain end functionality on the formed polymer segment. The polymers synthesized using ATRP show tolerance to many functional groups, such as hydroxy, amino, amido, esters, carboxylic acid, that can be incorporated into a copolymer then used for post-polymerization modifications including covalent linking of biomolecules and drug delivery. As disclosed below, this enables formation of bioconjugates between synthetic polymers and biomolecules. Thus, the delivery system synthesized using ATRP offer customizable and tunable structure for precise targeted delivery of biologically active molecules.
Methods for the incorporation of an unnatural amino acid by use of an orthogonal synthetase-tRNA pair wherein the gene for the synthetase is randomized for the codons corresponding to the desired active-site residues is discussed by Xie & Schultz. Alternating positive and negative selection on the resulting library of synthetases produces clones which are transformed into cells with a plasmid containing a gene interrupted with an amber codon (see Miyake-Stoner & Refakis et al., Biochemistry 2010, 49, 1667; Stokes et al., Molecular Biosystems 2009, 5, 1032).
Techniques for the characterization of the incorporation of an initiator into proteins in response to the amber codon are described by Miyake-Stoner & Refakis et al.; Stokes et al.; Miyake-Stoner & Miller et al., Biochemistry 2009, 48, 5953.
Despite the importance of protein-polymer bioconjugates, there is no general method for producing homogeneous recombinant proteins that contain polymer initiators at defined sites (see Broyer et al., J. Am. Chem. Soc. 2008, 130, 1041).